Amounting to the largest biomass on the planet, bacteriophages are seen to have a huge potential in the biopharmaceutical landscape. With the rapid emergence of antibiotic resistance in bacterial pathogens, research interest in phage therapy has grown. Controlling the lytic action of phages will allow for the development of an inducible phage therapy and will mitigate concerns over rapid toxin release.
We demonstrated an alternative method of making P2 phage lysate progeny involving washing of E. coli P2 infected cells with magnesium sulfate, providing higher titers of phage. We reported the effect of cleaving the P2 genome with the type II CRISPR-Cas9 system using a spacer targeting lysA, showing that P2 is not very susceptible to Cas9 nuclease attack with the specific spacer. Observing deviances in replication rates when P2 is subject to Cas9 nuclease attack. And develop a potential genome editing strategy involving the co-delivery of a CRISPR-cas9 plasmid and donor plasmid containing a sequence at interest. These results provide insight into some of the issues that may arise when attempting to edit phage genomes with CRISPR-Cas9.
1.0. Introduction
The gut microbiota is made up of a wide range of microorganisms including eukaryotic viruses, bacteria, fungi, archaea and bacteriophages. All these organisms work in conjunction with each other to digest dietary fiber and other polysaccharides to generate compounds (such as amino acids and sugars), which are then used to resupply cells in the colon wall. This microbiota in recent years has been shown to modulate metabolism, immune system and protect against carcinogenesis and pathogens [1]. Some specific bacterial strains have proved beneficial to the human host when administered in adequate amounts, being referred to as probiotics. The best example of this being E. coli Nissle 1917 (EcN) to which those with EcN in their gut microbiota were less likely to develop infectious diarrhea. At present EcN is the main active ingredient in the oral drug, Mutaflor, marketed by Ardeypharm GmbH and used for the treatment of diarrhea and IBD.
1.1. Phage therapy
In a time where the end of antibiotics could soon be a possibility with already up to 50,000 lives are lost every year in Europe due to multiantibiotic resistant infections [Wellcome trust], routine surgeries and minor infections will once again become life threatening and we will be thrown back 100 years of medical advancements and the CDC projects that if left unchecked deaths per year will rise to 8.2 million per year in 2050. Because of this, it is of the utmost importance that we always stay a step ahead of pathogens and develop new alternative treatments. Issues with current antibiotic treatments is that they are normally simple compounds that disrupt the normal functioning of the pathogen, such as penicillin which disrupts the formation of the peptidoglycan wall, causing the cell to shed their walls when the bacteria divide. The problem with this however is that this single compound is extremely easy for pathogens to develop resistance mechanisms to them over time, or in places like sewers resistant bacteria strains to supply resistance genes to other pathogens via horizontal gene transfer. Another problem with antibiotics is that they have very little specificity, mostly being limited to whether the bacterium is gram positive or negative. This causes many problems when stronger, broad spectrum antibiotics are used in hospitals, which end up killing all the “good” bacteria in the gut microbiota, leaving space for pathogens such as C. difficile to manifest itself in patients causing potentially life threatening inflammation of the colon. A solution to this constantly evolving problem is to create a therapy that constantly evolves itself to get around any resistance mechanisms bacteria may come up with against it and also one that is highly specific to target only the problematic bacteria. Fortunately, this already exists. Bacteriophages are a virus that infects bacteria, they undergo mutations and natural selection much like bacteria to get around any resistance mechanisms that may arise and are extremely specific to the single receptor they have complementarity to so can be targeted against only a single strain of bacteria.
Bacteriophages inhabit all waters on earth, likely constituting the largest total biomass on the planet [33]. Since their discovery in the early 20th century, phages have proved to be highly useful models to describe some of the basic mechanisms of life, creating new avenues for phage therapies and genetic engineering with the soviets expressing lots of interest during ww2 [8-10]. A French- Canadian Scientist at the Institute Pasteur and the co-discoverer of bacteriophages utilized cocktails of lytic phages to treat bacterial infections almost a century ago [9,10]. Despite this, the discovery of novel antibiotics which provided a greater potency and target range lead to therapeutic phage research lagging behind [11]. The recent emergence and spread of multiantibiotic resistant pathogens has recently revived efforts to develop new phage therapies. A compelling example being in April 2017 in San Diego, an individual became infection with multidrug resistant Acinetobacter baumannii, after being in a coma for nearly two months, intravenous administration of a cocktail of phages that infect and lyse Acinetobacter baumannii led to the individual make a complete recovery [15].
To make phage therapy available as an oral therapy, we plan to make probiotic bacteria lysogenic for phages against pathogenic gut bacteria such as C. difficile or E. coli O157:H7. The probiotic bacteria (most likely E. coli Nissle) will host the therapeutic phage, with the lytic phase of therapy to be inducible with IPTG (or some other small molecule) through using recombineering in a suitable lysogenic strain to put a phage-encoded gene under a lac promoter. This is to prevent endotoxin release if the cells are continuously lysing randomly. The lytic phase should not lyse all of the probiotic therapy in the gut at once however when induced with IPTG to allow phage to stay at therapeutic levels within the gut for long periods of time. This could be perhaps achieved through the natural occurrence of “biological noise”, used by cells and viruses to ensure the survival of a population through stochastic activation of pathways and gene expression [12-14].
This experiment is focused upon removing the lytic capability of the phage, specifically the gene lysA.
1.2. P2 Structure and Genome
Bacteriophages are viruses that attack specific bacteria, being observed in both animals and humans (whether that be oral, respiratory or gastrointestinal) [2]. These viruses are parasitic biological entities that require its host cell to provide the machinery and fuel for its replication. They are made up of a single or double stranded RNA or DNA, contained within a protein capsid, forming an overall structure called a virion. The overall structure of P2 shown in figure 1, P2 belongs to the Myoviridae family with a capsid and a contractile tail using these it replicates via:
1. Adsorption: phage attaches to target cell via tail fibers.
2. Ejection of viral DNA into host cytoplasm via contraction of tail sheath.
3. Transcription and translation of early genes.
4. Synthesis of linear concatemers copies of viral DNA through rolling circle. And then transcription and translation of late genes.
5. Assembly of an empty procapsid and packaging of genome
6. Viral tail fibers assembly and viral tail assembly
7. Mature virion’s are released from cell by lysis
The host range of P2 and a number of other bacteriophages is determined by the tail fibers, during the adsorption stage of the host, the tail fiber reversibly binds to a receptor on the surface of the bacterial cell, after this the irreversible steps of injection of phage DNA into the cell occurs and the host cell is now infected. However, after new P2 progeny have been assembled, this progeny needs to be released via lysis of the cell.
Lysis of the cell is carried out via the translation of lysis genes (Figure 2). lysA, lysB and lysC are all dispensable under certain conditions of growth. The lysA gene, although non essential functions as an antiholin, needed to delay the action of gp Y until optimal lysis time, amber mutants in lysA causes slightly accelerated lysis [4].
1.3. Type II CRISPR-Cas9
Classic genetic modification strategies are tedious, complex genome modifications such as cytosine hydroxylmethylation and glycosylation which makes DNA resistant to the majority of restriction endonucleases. Recently however, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system was developed, being an efficient tool for genome editing in many organisms [5]. CRISPR-Cas is an active immune system evolved in archaea and bacteria to counter the invasion of foreign genetic elements such as plasmids and bacteriophages [34,35]. Upon phage infecting a bacterium, the phage incorporates short 20 to 40 base pair segments of phage genome, known as spacers, into a CRISPR array in the bacterial genome. Surviving bacteria then use these acquired spacer sequences, expressing them as CRISPR RNA (crRNAs), when cells are infected with the same phage the crRNAs act as guides for the bacteria’s CRISPR-Cas system to the respective spacer sequence in the phage genome called the protospacer. CRISPR-Cas9 assembles and cleaves the phage genome, inactivating it and making it unable to produce progeny. Cleaved phage genome is cannibalized to procure additional spacers [35].
The type II CRISPR-Cas, originating from Streptococcus pyogenes, contains three components; Cas9 nuclease, guide RNA RNA derived from unique spacer sequences present in the CRISPR region and tracrRNA common to all spacers [5]. The guide RNA contains a sequence homologous to that of the target sequence to be cleaved. However, the target sequence must be followed by a protospacer adjacent motif (PAM), a 2-6 base pair DNA sequence to which is essential for Cas9 to bind to upon assembly. Expressed in a cell, these components assemble to form a CRISPR-Cas9 complex, creating a double strand DNA break at the target site in the genome that is complementary to the spacer sequence in the guide RNA. The break can then be later repaired, rejoined or recombined with processes such as homologous recombination to generate mutants of interest [5]. While this system has been extensively used in engineering mammalian genomes, there has been surprisingly little examples of CRISPR-Cas9 in engineering phage genomes [6].
1.4. Experiment Aims
The aim of this experiment was to engineer E. coli BW25113, using the CRISPR-Cas9 type II system, to prevent bacteriophage P2 from replicating with guide RNA targeting the lysA gene in the P2 bacteriophage genome to cut the genome and effectively kill the phage. Creating a lysate from this and performing plaque assays, we report the effectiveness of the CRISPR system in killing phage.
2.0 Methods
2.1. Bacterial and Bacteriophage strains
WT P2 Vir1, a lytic phage, was propagated on E. coli BW25113 at OD600 0.2-0.3. Phage stocks containing phage at a frequency ≈ 1 x 10-7 were initially used in the preparation of phage lysate.
2.2. Plasmids
CRISPR-Cas9 spacer plasmids were constructed with a chloramphenicol resistance cassette and tet promoter as shown in figure 6A and 6B.
Four standard biobrick-compatible goldengate cloning plasmids were built and used for generic goldengate cloning of our spacer sequences (Figure 5) according to Engler et al.
2.3. Preparation of P2vir1 lysate
The CRISPR-Cas9-Lysa-gRNA plasmids was transformed into E. coli BW25113, E. coli was also transformed with CRISPR-Cas9 off target plasmid without guide RNA. During the preparation of P2 lysate, two modified protocols were used. Both protocols were the same apart from 1) we grew our E. coli cultures to half of the exponential phase OD600 = 0.2-0.3 in LB medium with 2mM CaCl2, and then infected with phage P2vir1 with a multiplicity of infection (MOI) of 0.01 and incubating for 10 minutes at 37°C, then washing the cells 3 times with 10 mL of MgSO4 and resuspended in LB medium with 5mM CaCl2, 16mM MgCl2 and Glucose to 1% where it was left to incubate for 3 hours or until lysis occurred. 1mL of chloroform was added and incubated at room temperature for 30 minutes. The lysate was then centrifuged at 3600xg for 10 minutes and filtered to remove cell debris. 2) We grew our E. coli to OD600 = 0.9-1.2 in LB medium, and then infected with phage P2vir1 with a multiplicity of infection (MOI) of 0.01 and incubating for 10 minutes at 37°C. Without washing, 5mM CaCl2, 16mM MgCl2 and Glucose to 1% was added and left to incubate for 3 hours or until lysis occurred. 1mL of chloroform was added and incubated at room temperature for 30 minutes. The lysate was then centrifuged at 3600xg for 10 minutes and filtered to remove cell debris and did not wash the cells, carrying on with the protocol.
2.4. Plaque Assays
The efficiency of CRISPR-Cas and gRNA-lysA in restricting P2 phage infection was determined via plaque assay. E. coli in LB was cultured to an OD600= 0.2-0.3, 0.3mL of culture was added to 0.1mL serial dilutions of phage lysate. After incubation for 10 minutes, 3 ml of 0.5% top agar with 2 μL of CaCl2 was added into each tube and poured onto 5mM CaCl2 LB plates. pfu/mL was calculated.
3.0. Results
To determine if the WT P2 vir1 genome can be inactivated by CRISPR-Cas9 nuclease, we constructed 2 plasmids, with pCas9-LysA-gRNA containing the 25nt spacer sequence (Figure 7). If the gRNA-Cas9 complex is functional, it would cleave the P2 genome at LysA corresponding to the protospacer sequence. Thereupon the genome will be disrupted likely leading to the loss of plaque forming ability of the virion. On the other hand, if the genome is resistant to CRISPR-Cas9 cleavage, plaques will appear at a similar frequency as the control plasmid lacking lysA-guide RNA.
3.1. Optimized P2 lysate protocol
The plating efficiencies of WT P2 phage vir1 was determined for the CRISPR-Cas9 plasmid with lysA guide RNA, plasmid with only CRISPR-Cas9 and WT control BW25113. This was done by plaque assay (figure 4). Two slightly different protocols were used in the preparation of P2 lysate as described in methods. The effects of washing mid exponential phase E. coli BW25113 cells with MgSO4 is described in table 1. When comparing protocol 1 against protocol 2 in all accounts, protocol 1 gives much higher titers (~1010 vs ~104), showing that washing and growing culture to mid exponential phase is much more efficient when preparing P2 lysate.
3.2. P2 restriction by CRISPR-Cas9 lysA spacer
As shown on table 2, when grown on E. coli BW25113 using protocol 1 the phage gives higher efficiencies when compared to BW25113 transformed with pCas9-lysA. However, the difference is not even one log, with P>0.05 the data between all the results are not significant and we accept the null hypothesis and thus these data demonstrate the the P2 phage is not significantly restricted by CRISPR-Cas9 with a spacer targeting lysA. Suggesting two outcomes: Phage did not have time to absorb into cells, or that cutting was good, however mutations occurred allowing phage to evade Cas9 nuclease attack.
3.3. Plaque morphology
Looking closer at the plaque assays we observe a high frequency of smaller sized and much larger variance in size of plaques in lysate made from pCas9-lysA and pCas9-offtarget compared to control BW25113 cells which exhibit a relatively uniform size of plaques across cells. More time is needed to investigate this phenomenon, however it appears that we have a higher rate of phages that have a lower replication rate compared to BW25113 control. Potentially being caused by mutations occurring during normal P2 replication, this apparently higher mutation rate appears to be incurred
in the presence of CRISPR-Cas9.
4.0. Discussion
P2 phage is a well characterized virus, with all of the components of the virus from head to tail fibers and DNA packaging machines have been determined however much is still to be learned about them with significant knowledge gaps remaining [17]. The mechanisms of assembly being well known for P2; we can use this knowledge to engineer P2 to do as we will. With the recent development of CRISPR-Cas9 type II system, it has been easier than ever to modify genomes allowing the opening up of a platform to deliver genes and proteins therapeutically to mammalian cells in other phages [18-20], what learned here may be applied to other phages.
Our study found 2 main findings:
4.1. Better optimized P2 lysate protocol
During phage lysate production, growing our culture to mid exponential phase and washing with MgSO4 yields much higher viral titers. This is most likely due to the importance of metals in biology and their abundance in the earth, phages have evolved to use metals such magnesium. Many studies have recognized the requirement for magnesium as a cofactor as part of adsorption since the late 1950’s [21]. The importance of metals also extends to calcium chloride with reports for the importance of calcium chloride being described also in the 1950s [22,23]. Magnesium stimulates steps beyond adsorption, including the intracellular synthesis of phage progeny [23,24,25]. Metals are co-factors, contributing to the structure and maintenance of phage proteins [26]. So washing out the cells and supplying excess magnesium ions enhances the uptake and replication of phage P2 during lysate production to yield higher numbers. Another advantage of washing out cells with MgSO4 is through washing out the cells, any free, unabsorbed phages are removed so your final lysate is much more likely to contain only phage that has been subject to the CRISPR system inside your cell
4.2. pCas9-LysA restriction of phage P2 infection
We demonstrate that CRISPR-Cas9 cleavage of lysA is very inefficient with some but not a significant amount of killing of P2 by CRISPR-Cas9 nuclease attack. It appears that the vast majority of phage escapes CRISPR-Cas9 nuclease attack upon lysA, P2 phage genome is too small to contain dedicated defensive mechanisms against CRISPR-Cas9 such that some other prophage genomes contain [27-29]. A possible reason for this is phage did not have time to absorb into cells. This is highly unlikely as described before, any free unabsorbed phages will have been washed away during washing with MgSO4. Also, the phage pfu/ml counts (table 2) are way too high for the highly diluted original stock of phage added to make the lysate, being diluted by a factor of 150 and so pfu’s of pCas9-offtarget and lysA should be more than 150x lower than controls. So this negates this reason. The short time cycle of lytic phage may be another limitation in this method.
The sequence targeted for Cas9 nuclease attack (lysA) may not be a very efficient spacer for restricting phage replication. Figure 9 shows findings by a 2017 paper, for a wild type T4 phage efficiencies of Cas9 nuclease attack depends upon what point of the genome the guide RNA is targeted for [16]. Targeting our guide RNA at another essential gene may yield lower efficiencies of amplification.
Mutations may possibly have occurred allowing P2vir1 to escape Cas9 nuclease attack. We find that plaque assays featuring the presence of CRISPR-Cas9 during lysate preparation have a high abundance of smaller, variably sized plaques. In an organism with a high mutation rate [30], the selective pressure of the presence of CRISPR-Cas9 attempting to cleave away at P2’s genome may yield an increased rate of mutagenesis in its genome. Tao. P et al. (2017) finds that after sequencing plaques produced from WT T4 phage infections with the presence of CRISPR-Cas9 with spacer targets, most CRISPR-Cas9 escape phage plaques have mutations in the PAM trinucleotide sequence or protospacer sequence [16]. Therefore, the plaques in Figure 8C may have silent or semi-silent mutations in the protospacer or PAM region which has allowed these phages to evade cleavage. General opinion on CRISPR-Cas9 is that it is an adaptive immune system that has evolved to protect its bacterial host against phage infections, often being lethal [31]. However, another paper by Tao. P et al. (2018) tells that CRISPR-Cas9 may increase the rate of evolution of phage. They found that in phage progeny produced from CRISPR-Cas9 E.coli infected with WT T4 observe a mutation rate at about 6 orders of magnitude greater than controls. [32]
Plaques from Figure 8A, B and C were sent for sequencing to check for any mutations that may have allowed phage to escape CRISPR-Cas9 nuclease attack. However, delays from sequencing meant results could not be included in this paper in time, we are hypothesizing that surviving progeny from pCas9-lysA will have mutations in PAM and the protospacer sequence of lysA, as observed in Tao. P (2017).
4.3. Future experiments
Our studies have found a few interesting findings, however more research will need to be done to confirm and understand the significance of some of the findings.
Repeating this experiment with different spacer regions, perhaps targeting other lysis genes such as lysB or lysC that may hopefully yield higher Cas9 efficiencies.
As above, if P2vir1 genome is susceptible to CRISPR-Cas9 cleavage, it may be possible to edit the genome at the cleaved site. Using homologous recombination with a donor plasmid as shown in Figure 9, using a CRISPR-Cas9 spacer plasmid targeting a region that is more efficiently cut which that above mentioned experiment will tell us. Along with the CRISPR-Cas9 spacer plasmid, second plasmid containing an amber mutation in the PAM or protospacer region in the same E. coli which will make any phages whose genomes have successfully been cleaved and homologously recombined be resistant by CRISPR-Cas9 attack by spacers targeting those regions. Upon infection of these E. coli cells by P2 phage, delivered genome will be cleaved which will cause the silencing of the genome and loss of plaque forming capability. In spite of this however, since the second homologous recombination plasmid is present, recombination of the cleaved ends of P2 genome and the donor will result in the transfer of the amber mutation from donor to P2 genome, which will restore integrity of the genome and plaque forming capability. Due to time constraints, this could not be done in time for this paper. Higher cleavage efficiencies will have to be reached however, hopefully by changing the spacer sequence, to reduce noise when attempting to sequence plaques.
If this proves to be successful, this methodology could be applied to remove entire lysis genes or any other genes and replace them with a new sequence replication through using two adjacent spacers. The CRISPR-Cas9 complex may be directed to make two cuts on the P2 genome and the homologous arms on the donor plasmid may then replace the excised sequence with a different mutant sequence. This could potentially allow for the complete manipulation and engineering of P2 phage, further research can then be done to characterize and understand the components P2 and this methodology may be applied to phages better suited for therapeutic use.
4.4. Conclusion
In conclusion, our studies have established a better methodology for making P2 lysate. We have investigated, for the first time, the effect of our designed spacer in the CRISPR-Cas9 cleavage at lysA in P2vir1 genome in E. coli BW25113 cells, observing deviances in surviving progeny plaque sizes. Showing that the spacer targeting lysA did not significantly kill P2. We describe the potential reasons for this outcome and described potential improvement and potential applications of this methodology to other phage genomes to harness the therapeutic potential of phages.
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